The establishment of an intimate interaction between the long retinal pigment epithelium (RPE) microvilli and the mature photoreceptor outer segments is essential for vision. The RPE performs highly specialized, unique functions essential for homeostasis of the neural retina. These include phagocytosis of photoreceptors shed outer segments, directional transport of nutrients into and removal of waste products from photoreceptor cells and visual pigment transport and regeneration. The apical microvilli of the RPE play a key role in mediating these activities. The hypothesis of this proposal is that proteomic comparison of young and old apical microvilli of the retinal pigment epithelium (RPE) will provide insight into fundamental RPE functions specifically affected by aging. This proposal explores the concept that the RPE apical microvilli can be studied to understand key age-related changes taking place in the RPE. Experiments in aim 1 will use a basic protocol to obtain intact RPE microvilli from young and aged rats. State-of-the-art proteomic methodologies and several biochemical assays will determine the protein composition and significant post- translational modifications associated with the samples in each age group. Confocal and immunoelectron microscopy analysis will further quantify the changes in the expression of relevant proteins. Experiments in aim 2 will characterize the role of ezrin in the aged RPE microvilli. Ezrin is an actin-binding protein involved in processes such as cell senescence and signal transduction. In the RPE, we have shown that ezrin is key to the development of apical microvilli and basal infoldings. This research will therefore also pursue a comparison of ezrin binding-partners in young and aged RPE apical microvilli and -MV fraction to better understand senescence-related changes in this cell. We will also investigate whether a differential post-translational modification of ezrin in aged RPE microvilli is somehow correlated to the age-related decrease of RPE apical microvilli. The long-term goal of this research is to identify proteins specifically affected by normal and pathological aging processes. The decline in vision in elderly, a direct consequence of changes in the neuro-retina and RPE, is a well-documented phenomenon. This research project will characterize physiological RPE ageing and relate it to ocular age-related diseases.